Breast cancer remains the second leading cause of cancer death in women. Our goal is to control breast cancer with HER2/HER3 dual target DNA vaccines and by in situ immune priming to additional tumor- associated antigens (TAA). Over-expressed or amplified antigens common to many cancers are the most appropriate targets for cancer vaccines for the mass. We have been successful in generating humoral and cellular immunity with HER2 DNA vaccines in mice, cats and humans. New insight from domestic cat vaccination studies showed functional immunogenicity of single residue substituted HER2. Building on these positive findings, we will further vaccine efficacy and broaden the coverage by targeting both HER2 and HER3 with novel vaccine design. We will test the hypothesis that human-primate (hp) composite DNA vaccine phpHER2/hpHER3 with selected substitutions in primate polymorphic residues induces functional immunity in HER2/HER3 transgenic, Diversity Outbred (DO) mice that model after human genetic diversity. The unique genomic mosaics of each DO mouse will be precisely mapped based on the fully sequenced genomes of their original founders. Specific genes regulating vaccine response will be defined in HER2 /HER3 transgenic, DO mice by single nucleotide variant (SNV) linkage analysis. We will further test the hypothesis that priming to additional endogenous TAA following HER2/HER3 vaccination is achieved by albumin binding, lymph node (LN) seeking toll-like receptor (TLR) agonists and strategic cytokine modulation. Anti-tumor effector cells to HER2/HER3 and other TAA induced by systemic and local immunization will be expanded by IL-12 gene expression, superagonist IL-15 treatment, and blocking negative regulatory mechanisms. With the innovative and comprehensive approach, durable immunity against breast cancer is expected. Our specific aims are: Aim 1 Formulate and test phpHER2/hpHER3 designer DNA vaccines using HER2/HER3 transgenic inbred mice Aim 2 Test candidate vaccines in HER2/HER3 transgenic Diversity Outbred (DO) mice and analyze the genetic linkage of vaccine response Aim 3 Boost immunity to endogenous tumor-associated antigens (TAA) via local and systemic immune modulation.